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Nucleic Acids Research, 1988, Vol. 16, No. 14 6361-6372
© 1988

Articles

Total synthesis and expression of a gene for the {alpha}-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin)

Thomas P. Sakmar and H.Gobind Khorana

Departments of Biology and Chemistry, Massachusetts Institute of Technology Cambridge, MA 02139, USA

Received February 29, 1988. To facilitate structure-function studies by site-specific mutagenesis, we have synthesized a gene for the {alpha}-subunit of the bovine rod outer segment (ROS) guanine nucleotide-binding protein (transducin). The gene codes for the native amino acid sequence and contains, by design, 38 unique restriction sites which are uniformly spaced. This enables mutagenesis in any part of the gene by restriction fragment replacement. The gene is 1076 base pairs in length. It was constructed from 44 synthetic oligonucleotides which were joined enzymatically in vitro into four fragments which were cloned. The synthetic transducin gene and cDNA encoding transducin were expressed to similar levels in monkey kidney cells (COS-1) using a vector in which transcription was under the control of the adenovirus major late promoter.


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